Prediction of diabetes impaired wound healing by urinary nitrate assay

ABSTRACT

Wound healing is impaired in many diabetics, who suffer increased risk of chronic foot ulceration and amputation. Diabetic patients with poor healing ability were found to possess significantly lower fasting urinary nitrate levels than diabetic patients with normal healing ability or non-diabetic controls, implicating decreased endogenous nitric oxide activity as the mediator of diabetes-impaired wound healing. Methods and kits are provided for predicting the wound healing ability of diabetic patients based on the levels of nitric oxide related products such as nitrate or nitrite in urine or other specimens. Methods are also provided for treating non-wound healing diabetics and monitoring diabetic ulcer treatment.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/125,284, filed Mar. 19, 1999, which is herebyincorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] The invention is related to the area of wound healing indiabetes. In particular it is related to assays for the level of nitricoxide in non-healing diabetics.

BACKGROUND OF THE INVENTION

[0003] Diabetes affects an estimated 15 million people in the UnitedStates. Within the diabetic population are individuals with chronic,non-healing lower extremity ulceration (LEU), which is associated withsignificant morbidity and treatment costs. Chronic, non-healing LEUprecedes about 85% of the lower extremity amputations (LEA) that over50,000 diabetics experience annually (G E Reiber, E J Boyko, D G Smith,in Diabetes in America, NIH Publication No. 95-1468, Bethesda, Md., ed.2, 1995, pp. 409-428). This represents more than half of all individualsreceiving LEA in this country. While only 6% of diabetichospitalizations are associated with LEU, the total governmentreimbursement for diabetic lower extremity complications in 1992exceeded $1.5 billion, not including costs for limb amputation andrehabilitation. Clinical pathophysiologic risk factors for LEA includediabetic neuropathy, lower extremity ischemia, and chronic, non-healingdiabetic foot ulcers.

[0004] The underlying problem in diabetics with LEU is impaired woundhealing, which is poorly understood. While the majority of diabeticsexhibit “normal” wound healing, those presenting with chronic LEU oftendemonstrate decreased wound inflammation, recurrent wound infections,decreased cutaneous vascular perfusion, poor wound collagen deposition,and scar maturation. Platelet derived growth factor (PDGF) deficiency isassociated with the chronic diabetic ulcer and contributes to impairedhealing (H D Beer, M T Longaker, S Werner, J Invest Dermatol 109, 132(1997)). Clinical trials using Regranex® have shown efficacy inimproving chronic foot ulcer healing in only half or less of thepatients evaluated (D L Steed, J Vasc Surg, 21, 71 (1995)).

[0005] Recent research on the role of nitric oxide (NO) in woundinflammation, tissue repair, and microvascular homeostasis has allowedus to consider NO as a primary regulator of wound healing (DBruch-Gerharz, T Ruzicka, V Kolb-Bachofen. J Invest Dermatol. 110, 1(1998); M R Schaffer et al., Surgery 121, 513 (1997)). A systemicdeficiency of endothelial-derived NO has been observed in all diabetics(A Veves et al., Diabetes, 47, 457 (1998); M Huszka et al., ThrombosisRes, 86(2), 173 (1997); S B Williams, J A Cusco, M A Roddy, M THohnston, M A Creager, J. Am. Col. Cardiol., 27(3), 567 (1996)),suggesting that NO plays a fundamental role in the pathogenesis ofchronic, non-healing LEU. Consequently, there is a need to correlate NOproduction with wound healing ability in diabetics. Such a correlationwould allow the development of methods to predict the wound healingability of diabetics based on their production of NO and would provide auseful clinical indicator which could serve as a basis for choosingappropriate therapy.

SUMMARY OF THE INVENTION

[0006] It is an object of the invention to provide a method ofdetermining whether a diabetic subject is a wound healing diabetic or anon-wound healing diabetic. The method comprises the steps of: (a)collecting a specimen from the subject; (b) determining the level of anitric oxide related product in the specimen; and (c) comparing thelevel of the nitric oxide related product in the specimen with athreshold value which discriminates between wound healing and non-woundhealing diabetics. If the level of the nitric oxide related product isabove the threshold value, then the subject is a wound healing diabetic.If the level of the nitric oxide related product is approximately at orbelow the threshold value, then the subject is a non-wound healingdiabetic.

[0007] It is also an object of the invention to provide a method oftreating a diabetic subject. The method comprises the steps of: (a)collecting a specimen from the subject; (b) determining the level of anitric oxide related product in the specimen; and (c) comparing thelevel of the nitric oxide related product in the specimen with athreshold value which discriminates between wound healing and non-woundhealing diabetics; and (d) treating the subject according to whether thesubject is a wound healing diabetic or a non-wound healing diabetic.

[0008] It is a further object of the invention to provide a method ofmonitoring the effectiveness of treatment of a non-wound healingdiabetic patient. The method comprises the steps of: (a) administeringto the patient a therapeutic agent designed to raise the level of nitricoxide in the patient; (b) collecting a specimen from the subject; and(c) determining the level of a nitric oxide related product in thespecimen as a measure of the effectiveness of the treatment.

[0009] It is yet another object of the invention to provide a kit fordetermining whether a diabetic subject is a wound healing diabetic or anon-wound healing diabetic. The kit comprises reagents for determiningthe level of a nitric oxide related product in a specimen andinstructions for obtaining the specimen, using the reagents, andinterpreting the results.

[0010] These and other objects of the invention are provided by one ormore of the embodiments described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 presents a schematic representation of the role of nitricoxide (NO) in wound repair regulation. Wound NO mediated “cellularsignaling” appears to enhance the inflammatory mediation of repair,wound oxygen availability, and wound matrix remodeling and maturation.

[0012]FIG. 2 is a graphical representation of the fasting urine nitratelevels (micromoles per liter) for controls (C), healed diabetics (HD),and unhealed diabetics (UHD) on days 1 and 2 of the study. Results areshown as mean±S.E., with P-values as compared to C (Pc) and HD (PHD) foreach day.

[0013]FIG. 3 is a graphical representation of the fasting plasma nitratelevels (micromoles per liter) for controls (C), healed diabetics (HD),and unhealed diabetics (UHD) on days 1 and 2 of the study. Results areshown as mean±S.E., with P-values as compared to C for each day except‡, which compares HD and UHD for Day-2 only.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The methods and kits of the invention are designed to detect,treat, and monitor diabetic patients with poor wound healing abilitybased on measurement of the breakdown products of NO in specimens takenfrom the patient under controlled conditions. The invention presumesthat diabetic patients represent a continuous spectrum of NO syntheticcapability and that diabetics at the lower end of that spectrum haveimpaired wound healing function. NO is normally metabolized to thestable products nitrate and nitrite, which may be assayed in urine,plasma, tissue, or other specimens from diabetic patients. The level ofnitrate or nitrite in a specimen can serve as an indicator of the levelof NO synthesis in a patient. The findings of the inventor indicate thatbelow a threshold level of NO in the diabetic patient, normal woundrepair is not achieved, resulting in LEU.

[0015] NO is a small, hydrophobic gaseous free radical which is animportant physiological mediator for autonomic functions such asvasodilatation, neurotransmission, and intestinal peristalsis. NOprovides cellular signaling by activation of its target molecule,guanylate cyclase, which elevates intracellular concentrations of cyclicguanosine monophosphate (cGMP) (J S Beckman, in Nitric Oxide, J.Lancaster, Jr., Ed. (Academic Press, N.Y.), chap. 1). Cellular signalingis performed without mediation of channels or cellular membranereceptors and is dependent upon the concentration of NO in the cellularenvironment.

[0016] NO has a half-life of about five seconds in biological tissues.It is generated by three isoforms of nitric oxide synthase (NOS), whichmetabolize L-arginine and molecular oxygen to citrulline and NO. Two ofthe three isoforms are constitutive enzyme systems (cNOS) which aredescribed in neuronal cells (nNOS) and in endothelial cells (eNOS) (DBruch-Gerharz, T Ruzicka, V Kolb-Bachofen. J Invest Dermatol. 110, 1(1998)). With these isoforms, increased levels of intracellular calciumactivate the enzymes via calmodulin. The calcium-dependent cNOS systemsproduce low (picomolar) concentrations of NO. The third system is theinducible isoform (iNOS) which is calcium independent. The expression ofiNOS is induced by tissue-specific stimuli such as inflammatorycytokines or bacterial lipopolysaccharide (LPS). The inducible isoformreleases NO in much higher (nanomolar) concentrations than cNOS and haspotent cytotoxic effects.

[0017] Currently, it appears that the cNOS enzymes are involved inregulating and maintaining skin homeostasis (S Moncada, A Higgs, N Eng JMed 329, 2002 (1993)). The iNOS enzymes appear to be mainly associatedwith inflammatory and immune responses that are also implicated incertain skin diseases. In human skin keratinocytes, fibroblasts andendothelial cells possess both the cNOS and iNOS isoforms. The woundmacrophage and keratinocyte possess the iNOS isoform. In wound healingstudies NO synthesis has been shown to occur for prolonged periods(10-14 days) after wounding and macrophages appear to be the majorcellular source (M R Schaffer, U Tantry, R A vanWesep, A Barbul. J SurgRes, 71, 25 (1997)). As a mediator of tissue repair, NO has beendemonstrated to promote angiogenesis (A Papapetropoulos, GGarcia-Cardena, J A A Madri, W C Sissa. J Clin Invest, 100(12), 3131(1997)) and cellular migration (E Noiri et al., Am. J. Physiol. 279:C794(1996)), increase wound collagen deposition and collagen cross-linking(M R Schaffer, U Tantry, S S Gross, H L Wasserburg, A Barbul. J SurgRes, 63, 237 (1996)), regulate microvascular homeostasis(vasodilatation) (D Bruch-Gerharz, T Ruzicka, V Kolb-Bachofen. J InvestDermatol. 110, 1 (1998)), inhibit platelet aggregation (J S Beckman, inNitric Oxide, J. Lancaster, Jr., Ed. (Academic Press, N.Y.), chap. 1),inhibit the formation of endothelial-leucocyte adhesions (A M Lefer, D JLefer, Cardiovascular Res. 32, 743 (1996)), modulate endothelialproliferation and apoptosis (Y H Shen, X L Wang, D E Wilcken, FEBS Lett,433(1-2), 125 (1998)), increase the viability of random cutaneous flaps(S C Um et al., Plast Reconstr Surg. 101 785 (1998); G F Pierce et al.,Proc Natl Acad Sci USA. 86, 2229 (1989)), and enhance cellularimmunomodulation and bacterial cytotoxicity (J S Beckman, in NitricOxide, J. Lancaster, Jr., Ed. (Academic Press, N.Y.), chap. 1).

[0018] In diabetics normal wound repair can be significantlycompromised. In general, during the wound healing process, NO providesenhancement of tissue oxygen availability, the inflammatory mediation ofrepair mechanisms and wound matrix development and remodeling (FIG. 1).The major metabolic pathway for NO is to nitrate and nitrite, which arestable metabolites within tissue, plasma, and urine (S Moncada, A Higgs,N Eng J Med 329, 2002 (1993)). Tracer studies in humans havedemonstrated that perhaps 50% of the total body nitrate/nitriteoriginates from the substrate for NO synthesis, L-arginine (P M Rhodes,A M Leone, P L Francis, A D Struthers, S Moncada, Biomed Biophys Res.Commun. 209, 590 (1995); L. Castillo et al., Proc Natl Acad Sci USA 90,193 (1993). Although nitrate and nitrite are not measures ofbiologically active NO, plasma and urine samples obtained from subjectsafter a suitable period of fasting, and optionally after administrationof a controlled diet (low nitrate/low arginine), allow the use ofnitrate and nitrite as an index of NO activity (C Baylis, P Vallance,Curr Opin Nephrol Hypertens 7, 59 (1998)).

[0019] The invention provides a method of determining whether a diabeticsubject is a wound healing diabetic or a non-wound healing diabetic. A“wound healing diabetic” refers to a diabetic subject whose woundhealing capability is approximately the same as that of a non-diabeticsubject. A “non-wound healing diabetic” refers to a diabetic subjectwhose wound healing capability is reduced from that of a non-diabeticsubject and who consequently is at risk for LEU. For example, in oneclinical study, non-wound healing diabetics were considered to be thosepatients with a history of one or more diabetic foot ulcers withincomplete healing after 20 weeks of Regranex® treatment (see Example1). A subject according to the invention can be any human or animal witha diabetic condition such as diabetes mellitus. A human or animal with adiabetic condition is a human or animal whose regulation of plasmaglucose concentration is defective, usually as a result of insufficientproduction of insulin or resistance to the physiological effects ofinsulin. For example, the subject can be a human patient who isdiagnosed by a physician as having either type I or type II diabetes.

[0020] One method of determining whether a diabetic subject is a woundhealing or non-wound healing diabetic comprises the steps of collectinga specimen from a diabetic subject, determining the level of nitrate ornitrite in the specimen, and comparing the level of nitrate or nitritein the specimen with a threshold value that discriminates between woundhealing and non-wound healing diabetics. In addition to nitrite andnitrate, other molecular species related to NO synthesis or breakdown(other “NO-related products”) can be quantified in blood, urine, tissue,or other samples from a patient. For example, plasma levels ofL-citrulline, which is a product of the reaction that produces NO, orcGMP, which is produced as a result of NO activation of guanylatecyclase, can be determined as a reflection of systemic NO synthesis in apatient. (F L Kiechle and T Malinski, Ann. Clin. Lab. Sci. 26,501(1996)). Similarly, L-dimethylarginine, another product of NOS, canbe detected by HPLC in human serum and used as a highly specific indexof systemic NOS activity. (J Meyer et al., Anal. Biochem. 247, 11(1997)). NO can also break down by reacting with superoxide anion inhuman plasma to produce peroxynitrite, which in turn can produce avariety of radicals such as ascorbyl radical and albumin-thinyl radicalthat can be detected using electron paramagnetic resonance (EPR)spectroscopy. (J Vasquez-Vivar et al., Biochem. J. 314, 869 (1996)).Another product of peroxynitrite is 3-nitrotyrosine, which can bedetected in human plasma or other fluids by gas chromatography in tandemwith mass spectrometry (E Schwedhelm et al., Anal. Biochem. 276, 195(1999)), reversed-phase HPLC (H Ohshima et al., Nitric Oxide 3, 132(1999)), or an ELISA method using anti-nitrotyrosine antibodies (J C terSteege et al., Free Radic. Biol. Med. 25, 953 (1998)). Unlike nitrate ornitrite, most of these products are not subject to interference bydietary intake. Furthermore, the in situ detection of NO itself ispossible with the aid of biosensors that quantify NO levels and changesin NO levels in response to stimuli. For example, the heme domain ofsoluble guanylate cyclase, a natural receptor for NO, can be labeledwith a fluorescent reporter dye, and changes in fluorescence intentitycan be determined through an optical fiber and calibrated to reveal NOlevels at any desired location in the body, for example at or near awound site (S L Barker et al., Anal. Chem. 71, 2071 (1999)). Given therapid decomposition of NO in biological fluids, direct detection of NOshould be performed in situ rather than some time following collectionof a specimen.

[0021] The specimen can be any sample of fluid or tissue obtained fromthe subject in sufficient amount as to allow the determination of thelevel of nitrate, nitrite, or other NO-related product. For example, thespecimen can be a sample of urine, blood (including plasma), or tissue.The specimen is preferably obtained from the subject after a period offasting, in order to allow the level of nitrate, nitrite, or otherNO-related products to achieve a stable baseline level. The period offasting reduces interference from dietary and metabolic sources ofnitrate or nitrite that are not related to NO breakdown. During theperiod of fasting, the subject's consumption of all solid and liquidfood is reduced from his average consumption by at least 50%, 60%, 70%,80%, 90%, or 100%. Preferably the subject's consumption of all solid andliquid food is reduced by at least 90%. More preferably the subject'sconsumption of all solid and liquid food is reduced by 100%. Mostpreferably, the subject does not consume any solid or liquid food duringthe fasting period. The subject's consumption of water generally is notrestricted during the fasting period; however in some embodiments, thesubject also consumes no water during the fasting period. The fastingperiod should be of sufficient duration as to allow a stable baseline tobe achieved in whatever parameter is to be measured. A stable baselineis the condition in which the parameter measured, e.g., urinary nitrate,is generally reproducible and not subject to large fluctuations betweenrepeated measurements or undue interference from dietary, metabolic, orother sources that are not related to NO metabolism. Preferably theperiod of fasting is at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, or 24hours. More preferably the period of fasting is from 4 to 12 hours, orfrom 6 to 10 hours, or from 8 to 10 hours, or from 10 to 12 hours.

[0022] It is understood that any requirement for fasting will dependupon which NO-related product is being quantified, because some suchproducts are hardly affected by diet; others may require only a brieffast. For example, plasma L-dimethylarginine (J Meyer et al., Anal.Biochem. 247, 11 (1997)) is unaffected by diet, whereas urinarynitrate/creatinine ratios are unaffected by diet if an overnight fast isperformed prior to collecting the specimen (P S Grabowski et al.,Arthritis Rheum. 39, 643 (1996)).

[0023] In some embodiments, the period of fasting is immediatelypreceded by a period during which the subject is administered a dietthat is sufficiently low in sources of dietary nitrate or nitrite toachieve a stable baseline value of whichever NO metabolite will bedetermined in the specimen. For example, the diet can be one from whichall vegetables and nitrate- or nitrite-preserved foods have beeneliminated. The diet can also have a reduced level of L-argininecompared to the subject's normal diet. For example, one diet provides alevel of nitrate of less than 900 mg/kg body weight/day, and a level ofnitrite of less than 9 mg/kg body weight/day.

[0024] The level of nitrate or nitrite in the specimen can be quantifiedby any method known in the art which provides adequate sensitivity andreproducibility. For example, the Griess reaction is aspectrophotometric assay for nitrate which can provide sensitivedetermination of nitrate and nitrite in biological fluid samples (MMarzinzig et al., Nitric Oxide 1, 177 (1997)). If the Griess reaction oranother nitrite assay is performed both with and without reduction ofnitrate to nitrite, then nitrate values can be obtained as thedifference between the nitrite values obtained for the reduced sampleand the non-reduced sample. The Griess assay can be made more sensitiveif a fluorescent product is obtained, e.g., by reacting nitrite with2,3-diaminonaphalene (T P Misko et al., Anal. Biochem. 214, 11 (1993)).Highly sensitive assays are also available which first reduce nitriteand nitrate (R S Braman and S A Hendrix, Anal. Chem. 61, 2715 (1989)) orany NO-related compound (M Sonoda et al., Anal. Biochem. 247, 417 (1997)to NO for detection with specific chemiluminesence reagents. A varietyof protocols have also been described for detecting and quantifyingnitrite and nitrate levels in biological fluids by ion chromatography(e.g., S A Everett et al., J. Chromatogr. 706, 437 (1995); J M Monaghanet al., J. Chromatogr. 770, 143 (1997)), high-performance liquidchromatography (e.g., M Kelm et al., Cardiovasc. Res. 41, 765 (1999)),and capillary electrophoresis (M A Friedberg et al., J. Chromatogr. 781,491 (1997)).

[0025] The “level” of nitrate, nitrite, or other NO-related productusually refers to the concentration (in moles per liter, micromoles perliter, or other suitable units) of nitrate or nitrite in the specimen,or in the fluid portion of the specimen. However, other units of measurecan also be used to express the level of nitrate or nitrite. Forexample, an absolute amount (in micrograms, milligrams, nanomoles,moles, or other suitable units) can be used, particularly if the amountrefers back to a constant amount (e.g., grams, kilograms, milliliters,liters, or other suitable units) of the specimens under consideration. Anumber of commercially available kits can be used. One such kit isdescribed in Example 2.

[0026] The specimen can be processed prior to determination of nitrateor nitrite as required by the quantification method, or in order toimprove the results, or for the convenience of the investigator. Forexample, processing can involve centrifuging, filtering, or homogenizingthe sample. If the sample is whole blood, the blood can be centrifugedto remove cells and the nitrate or nitrite assay performed on the plasmaor serum fraction. If the sample is tissue, the tissue can be dispersedor homogenized by any method known in the art prior to determination ofnitrate or nitrite. It may be preferable to remove cells and otherdebris by centrifugation or another method and to determine the nitrateor nitrite level using only the fluid portion of the sample, or theextracellular fluid fraction of the sample. The sample can also bepreserved for later determination, for example by freezing of urine orplasma samples. When appropriate, additives may be introduced into thespecimen to preserve or improve its characteristics for use in thenitrate or nitrite assay.

[0027] The threshold value of nitrate, nitrite, or other NO-relatedproduct can be determined by comparing diabetic subjects with normal andpoor wound healing ability using the method of detection describedabove. For example, by comparing the urinary nitrate levels of a groupof non-wound healing diabetics with a group of wound healing diabeticsor normal subjects, preferably following the administration of a lownitrate diet and after a fasting period, the nitrate levels of the twogroups can be compared as in Example 2. The threshold value can beselected from the data obtained. For example, the threshold can bechosen as a value slightly higher than the mean of the urinary nitratelevel of the non-wound healing group; the threshold value should bechosen such that the urinary nitrate levels of at least 70%, 80%, 90%,95%, 98%, or 99% of the non-wound healing diabetics tested would fall ator below the threshold. For human patients, the threshold value fornitrate in urine is between 15 and 50 micromolar. Preferably, thethreshold value for nitrate in human urine is between 20 and 45micromolar, or between 25 and 40 micromolar. More preferably, thethreshold value for nitrate in human urine is 20, 25, 28, 30, 32, 35,37, or 40 micromolar. For human patients, the threshold value fornitrate in plasma is between 2 and 20 micromolar. Preferably, thethreshold value for nitrate in human plasma is between 3 and 17micromolar, or between 4 and 16 micromolar. More preferably, thethreshold value for nitrate in human plasma is 2, 4, 6, 8, 10, 12, 14,16, 18, or 20 micromolar. When selecting a threshold value of nitrate,nitrite, or other NO-related product for use with a given type ofspecimen, for example human urine or plasma, it should be noted that theuse of different assays or methods of standardization could shift thenumerical ranges from those provided here.

[0028] Another embodiment of the invention is a method for treating adiabetic subject. In order to practice this embodiment, a subject isfirst identified as either a wound healing diabetic or a non-woundhealing diabetic by the method described above, or by another method.Since non-wound healing diabetics, as identified by the invention,suffer from reduced NO activity, they can be treated by any therapywhich is designed to increase NO production. Such therapies include, butare not limited to, administration of L-arginine through increasing itspresence in the diet, oral administration of a dietary supplementcomprising L-arginine in any pharmaceutically acceptable form, orparenteral or intravenous injection of a pharmaceutically acceptablepreparation comprising L-arginine. The dosage can be selected from anyprotocol known in the art which is designed to increase NO production inthe patient. A further therapy which increases NO production ishyperbaric oxygen therapy. Optionally, the administration of any therapydesigned to increase NO production in a subject can be combined with themethod described below to monitor the effectiveness of the therapy inenhancing NO levels in the subject. If the subject is found to be awound healing diabetic, the preferred treatment does not involve atherapeutic agent designed to increase NO production in the subject. Inthe case of a wound healing diabetic subject, a different type oftherapy can be employed. For example, administration of PDGF (Regranex®,or another PDGF preparation) can be effective to promote wound healingin a wound healing diabetic subject.

[0029] The invention can also be used to avoid therapies which may bedisadvantageous for certain diabetic patients. Any negative therapeuticinfluence on the synthesis of nitric oxide or its effectiveness inpromoting wound healing can be minimized through the use of theinvention. For example, glucocorticoid drugs are sometimes administeredto diabetic patients with LEU for their anti-inflammatory effect.However, glucocorticoids are known to selectively inhibit the expressionof iNOS (M W Radomski, R M Palmer, S Moncada, Proc Natl Acad Sci USA 87,10043 (1990)), and have been shown to decrease the amount ofnitrite/nitrate in wound fluid (A E Ulland, J D Shearer, M D Caldwell, JSurg Res 70, 84 (1997). For patients identified as non-wound healingdiabetics, whose NO synthetic capability is expected to be reducedcompared with wound healing diabetics, the use of steroids that wouldfurther suppress NO levels in the patient is undesirable. Thus,according to one embodiment of the invention, a patient identified as anon-wound healing diabetic is not treated with glucocorticoids or otherdrugs suspected to reduce NO levels in the patient.

[0030] In a different embodiment, the invention can be used as a methodof monitoring the effectiveness of treatment of a non-wound healingdiabetic. In this embodiment, a patient is undergoing treatment thatinvolves administration of a therapeutic agent designed to raise thelevel of nitric oxide in the patient. Examples of such therapeuticagents include the L-arginine and hyperbaric oxygen treatments describedabove. Following administration of the therapeutic agent, the patient ismonitored for effectiveness of the treatment by the method of detectingnitrate or nitrite in a specimen from the patient, as described above.If the nitrate, nitrite, or other NO-related product level in thespecimen is at or below the threshold value for determining whether thepatient is a wound healing or non-wound healing diabetic, then theeffectiveness of the therapeutic agent is insufficient to promote woundhealing. In that case, the treatment can be subsequently adjusted, forexample by increasing the dose or potency of the therapeutic agent orincreasing the period of exposure to the therapeutic agent. In a relatedembodiment, the method of monitoring the patient is repeated, and thedose or potency of the therapeutic agent, or period of exposure to thetherapeutic agent, is again increased. Preferably, in this embodimentthe method of monitoring and increasing the dose of the therapeuticagent is increased until the nitrate or nitrite level in a specimen fromthe patient is above the threshold value. It may be desirable to thenmaintain the therapy at the most effective dose as long as needed untilthe wounds of the patient have healed.

[0031] Yet another embodiment is a kit for determining whether adiabetic subject is a wound healing diabetic or a non-wound healingdiabetic. The kit comprises one or more reagents for determining thenitrate or nitrite level in a specimen from a diabetic subject. Thereagent or reagents can be those required by any method known in the artfor determination of nitrate or nitrite in a specimen. The kit alsoincludes a set of instructions for using the reagents to carry out themethod of determining whether a diabetic subject is a wound healingdiabetic or a non-wound healing diabetic, as described above. Theinstruction set provides information in any suitable format (e.g.,printed on paper or in electronic format on a diskette, CD-ROM, or byreference to a web site or printed publication) to allow the user tocollect a suitable specimen, process the specimen, use the reagent orreagents to determine the level of nitrate, nitrite, or other NO-relatedproduct in the specimen, and interpret the results obtained, i.e., todetermine whether the subject is a wound healing diabetic or a non-woundhealing diabetic.

[0032] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples, which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLE1 Specimen Collection from Wound Healing and Non-Wound HealingDiabetic Patients

[0033] To explore the hypothesis that non-wound healing diabetics haveimpaired NO activity, the following study of plasma and urinary NOmetabolites—nitrate and nitrite—was carried out following a diabeticulcer wound healing study using Regranex®. The results indicate that thechronic, non-healing LEU diabetic population is characterized bysignificantly decreased urinary nitrate excretion.

[0034] For the clinical study, ten (10) healthy, diabetic patientspresenting with a history of one or more diabetic foot ulcers werechosen. All patients had previously received topical ulcer treatmentwith Regranex® gel under close clinical observation. Half of this group(n=5) experienced complete healing (healed diabetics/HD) of the ulcer byweek 20 of observation. The remaining half (n=5) of this group had notexperienced complete healing (unhealed diabetics/UHD) of the ulcer byweek 20 of observation. Following the completion of Regranex® treatment,the 10 diabetic subjects (HD and UHD) and 10 healthy, non-diabeticcontrols (C) were enrolled for urine and plasma nitrate/nitriteanalysis. Prior to this analysis all subjects were screened with amedical history, physical examination, and baseline hematology, serum,and urine chemistry in order to eliminate subjects with active malignantdisease, rheumatic or collagen vascular disease, chronic renalinsufficiency, inflammatory bowel disease, alcohol/drug abuse,cellulitis, osteomyelitis and those requiring revascularization surgery.For compliancy reasons, subjects determined to exhibit poor diabeticcontrol were disqualified. Additionally, anyone receiving radiationtherapy, systemic corticosteroids, and immunosuppresive orchemotherapeutic agents was disqualified. Informed consent was obtainedfrom all study participants.

[0035] Subjects from all groups were brought into the hospitalenvironment after having fasted for a 10-hour period. Fasting urine andplasma samples were obtained from each subject upon admission (Day-1) toprovide an indication of the subject's baseline nitrate and nitritelevels. Additionally, routine labwork consisting of chemistry panel,CBC, and urinalysis was obtained from all subjects upon hospitaladmission. Subjects were confined to the hospital setting for 24 hours,during which time activity level, dietary intake, and otherenvironmental factors were controlled. All subjects were restricted tobed rest with bathroom privileges and consumed the same diet during the24 hr hospitalization (2,581 Kcals; 124.2 g protein; 5,779 mg arginine;see Example 3). All subjects were required to refrain from smoking andalcohol consumption. Vegetables that usually have a higher nitratecontent from fertilizers and nitrate- and nitrite-preserved foods wereeliminated from the study diet, which is shown in Table 1 below.Concomitant baseline medications were administered and blood glucosemonitoring was performed by the diabetic subjects per their usual homeroutine. Medication and dietary intake as well as urinary output wererecorded and evaluated by the research team during the 24-hourconfinement period. At 9 p.m. on the day of confinement, all subjectswere required to begin another 10-hour fasting period. Prior todischarge from the hospital setting, the subjects again provided fastingplasma and urine samples (Day-2). Vital signs were monitored dailyduring confinement and all subjects were evaluated for adverse eventsprior to discharge. All obtained plasma and urine samples wereimmediately frozen at −20° C. in preparation for laboratory analysis.TABLE 1 Research Diet Menu Calories Protein (g) Arginine (mg) BREAKFASTegg, 1 77 6.3 377 cereal, ¾ cold 80 3.0 240 toast, 1 slice, while 61 2.2n/a margarine, 1 tsp. 45 0  1 jelly, 1 tbsp. 50 0 n/a orange juice, 4oz. 55 0 112 milk, 2%, 8 oz. 121  8.1 294 LUNCH hamburger, 3.5 oz. 274 26.6 1615  bun 122  4.4 n/a ½ sliced tomato 15 .5  8 mayonnaise, 1 tbsp.100  0  10 corn chips, 1 oz. 153  1.9  92 mixed fruit cup 80 0 n/a milk,2%, 8 oz. 121  8.1 294 DINNER baked chicken breast, 3.5 oz. 222  29.01811  rice, white, ½ c. 133  4.9 342 apples, canned, ½ c. 68 0  6 dinnerroll 85 2.4 n/a margarine, 1 tsp. 45 0  1 cantaloupe cubes, ½ c. 28 1.4n/a diet pudding, ½ c. 250  6.2 n/a milk, 2%, 8 oz. 121  8.1 294

EXAMPLE 2 Determination of Urine and Plasma Nitrate and NitriteConcentrations in Wound Healing and Non-Wound Healing Diabetic Patients

[0036] Specimens of urine and blood were obtained from the wound healingand non-wound healing diabetic subjects as described in Example 1.

[0037] Urine and plasma samples were fluorometrically assayed fornitrite and nitrate levels using a commercial kit (Cayman Chemical, AnnArbor, Mich.) according to the manufacturer's instructions. The methodused is that described by Gilliam et al. (Anal. Biochem. 212, 359(1993)). Blood was collected in a glass tube, centrifuged, and theplasma collected and frozen until assay. The samples were thawed,vortexed and filtered with a 10 kDa size exclusion filter (Millipore,Bedford, Mass.). For the determination of nitrate, nitrate reductase andNADP was added and allowed to incubate at room temperature for twohours. Following incubation 2,3-diaminonapthalene followed by NaOH wasadded and the fluorescence determined with a fluorimeter usingexcitation at 365 nm and emission at 405 nm. Nitrite concentration wasdetermined using the same method with the exception that nitratereduction steps were omitted. Urine was processed in a similar fashionexcept that filtration was omitted. All samples were assayed intriplicate. Concentration in patient samples (micromoles per liter) wasdetermined by comparison to standard nitrate and nitrite solutions.One-way ANOVA with Tukey-Kramer post test (Ludbrook, Clin. Exp.Pharmacol. Physiol. 18, 379 (1991)) was performed using GraphPad InStatsoftware, version 4.10 for Windows 98. P-values <0.05 were consideredsignificant.

[0038] On Day-1 fasting urine nitrate levels (micromoles/l±S.E.) forgroups C and HD (55.88±4.49 and 54.14±3.32, respectively) were notsignificantly different (FIG. 2). However, group UHD fasting urinenitrate levels (30.35±3.61) were significantly lower than groups C(p<0.001) or HD (p<0.01). Day-2 fasting urine nitrate levels for groupsC and HD were lower (42.60±1.92 and 45.57±5.10, respectively) but againnot significantly different. Similarly, group UHD fasting urine nitratelevels (22.74±3.13) were lower than Day-1 values and were againsignificantly lower than either group C (p<0.05) or HD (p<0.05) [Table2]. Day-1 fasting plasma nitrate levels (micromoles/l±S.E.) for group C(4.80±0.85) and group UHD (4.05±0.37) were not significantly different(FIG. 3). Group HD (11.71±2.08), however, was higher than UHD, but onlysignificantly higher than C (p<0.05). Day-2 fasting plasma nitratelevels were slightly lower for groups C (2.92±0.37) and UHD (3.16±0.61),but as before these were not significantly different. However, Group HD(11.94±4.46) was now significantly higher that either group C (p<0.01)or UHD (p<0.05). Urine and plasma nitrate levels were approximately 100times greater than nitrite and were occasionally undetectable by thismethodology. For these reasons urine and plasma nitrite levels are notreported. TABLE 2 Fasting Urine and Plasma Nitrate Values DAY 1 DAY 2 CHD UHD C HD UHD N = 10 N = 5 N = 5 N = 10 N = 5 N = 5 Fasting 55.88 ±54.14 ± 30.35 ± 42.60 ± 45.57 ± 22.74 ± Urine 4.49 3.32 3.61 1.92 5.103.13 Nitrate* (μm/l) P †, ‡ †NS †.001 ‡NS ‡.05 Fasting 4.80 ± 11.71 ±4.05 ± 2.92 ± 11.94 ± 3.16 ± Plasma 0.85 2.08 0.37 0.37 4.46 0.61Nitrate* (μm/l) P †, ‡ †0.05 †NS ‡0.01 ‡NS §0.05

I claim:
 1. A method of determining whether a diabetic subject is awound healing diabetic or a non-wound healing diabetic, comprising thestep of: comparing the level of a nitric oxide-related product in aspecimen from the diabetic subject with a threshold value whichdiscriminates between wound healing and non-wound healing diabetics,wherein if the level of the nitric oxide-related product is above thethreshold value the subject is a wound healing diabetic, and if thelevel of the nitric oxide-related product is approximately at or belowthe threshold value the subject is a non-wound healing diabetic.
 2. Themethod of claim 1 , further comprising the step of: determining thelevel of a nitric oxide-related product in the specimen.
 3. The methodof claim 2 , further comprising the step of: collecting a specimen fromthe subject.
 4. The method of claim 3 , wherein the specimen iscollected following a fasting period of at least 6 hours.
 5. The methodof claim 4 , wherein the fasting period is from 6 to 10 hours.
 6. Themethod of claim 4 , further comprising the step of: administering to thesubject for at least 6 hours immediately prior to the fasting period adiet with an intake of nitrate below 900 mg/kg body weight/day and anintake of nitrite below 9 mg/kg body weight/day.
 7. The method of claim6 , wherein the diet is administered for a period of from 6 to 24 hoursin duration.
 8. The method of claim 1 , wherein the specimen is urine,blood, or tissue.
 9. The method of claim 1 , wherein the subject is ahuman.
 10. The method of claim 1 , wherein the nitric oxide-relatedproduct is nitrate, a derivative of nitrate, nitrite, or a derivative ofnitrite.
 11. The method of claim 10 , wherein the specimen is urine andthe threshold value is between 15 and 50 micromolar nitrate.
 12. Themethod of claim 11 , wherein threshold value is between 20 and 45micromolar nitrate.
 13. The method of claim 12 , wherein the thresholdvalue is between 25 and 40 micromolar nitrate.
 14. The method of claim13 , wherein the threshold value is about 35 micromolar nitrate.
 15. Themethod of claim 10 , wherein the specimen is blood and the thresholdvalue is between 2 and 20 micromolar nitrate.
 16. The method of claim 15, wherein the threshold value is between 3 and 17 micromolar nitrate.17. The method of claim 16 , wherein the threshold value is between 4and 16 micromolar nitrate.
 18. The method of claim 17 , wherein thethreshold value is about 5 micromolar nitrate.
 19. The method of claim 1, wherein the nitric oxide-related product is nitric oxide,L-citrulline, cGMP, peroxynitrite, 3 -nitrotyrosine, orL-dimethylarginine.
 20. A method of treating a diabetic subject,comprising the steps of: comparing the level of a nitric oxide-relatedproduct in a specimen from the subject with a threshold value whichdiscriminates between wound healing and non-wound healing diabetics,wherein if the level of the nitric oxide-related product is above thethreshold value the subject is a wound healing diabetic, and if thelevel of the nitric oxide-related product is approximately at or belowthe threshold value the subject is a non-wound healing diabetic; andtreating the subject according to whether the subject is a wound healingdiabetic or a non-wound healing diabetic.
 21. The method of claim 20 ,further comprising the step of: determining the level of a nitricoxide-related product in the specimen.
 22. The method of claim 21 ,further comprising the step of: collecting a specimen from the subject.23. The method of claim 22 , wherein the specimen is collected followinga period of fasting of at least 6 hours.
 24. The method of claim 23 ,wherein the fasting period is from 6 to 10 hours.
 25. The method ofclaim 23 , further comprising the step of: administering to the subjectfor at least 6 hours prior to the fasting period a special diet with anintake of nitrate below 900 mg/kg body weight/day and an intake ofnitrite below 9 mg/kg body weight/day.
 26. The method of claim 25 ,wherein the diet is administered for a period of from 6 to 24 hours induration.
 27. The method of claim 20 , wherein if the subject is anon-wound healing diabetic, the step of treating comprises administeringL-arginine to the subject at a therapeutically effective dose orperforming hyperbaric oxygen therapy on the subject.
 28. The method ofclaim 20 , wherein if the subject is a wound healing diabetic, the stepof treating comprises the administration of platelet-derived growthfactor to the patient at a therapeutically effective dose.
 29. Themethod of claim 20 , wherein the nitric oxide-related product isnitrate, a derivative of nitrate, nitrite, or a derivative of nitrite.30. The method of claim 20 , wherein the nitric oxide-related product isnitric oxide, L-citrulline, cGMP, peroxynitrite, 3-nitrotyrosine, orL-dimethylarginine.
 31. A method of monitoring the effectiveness oftreatment of a non-wound healing diabetic patient, comprising the stepsof: (a) administering to the patient a therapeutic agent designed toraise the level of nitric oxide in the patient; and (b) determining thelevel of a nitric oxide-related product in a specimen from the patientas a measure of the effectiveness of the treatment.
 32. The method ofclaim 31 , wherein the nitric oxide-related product is nitrate, aderiviative of nitrate, nitrite, or a derivative of nitrite.
 33. Themethod of claim 31 , wherein the nitric oxide-related product is nitricoxide, L-citrulline, cGMP, peroxynitrite, 3-nitrotyrosine, orL-dimethylarginine.
 34. The method of claim 31 , further comprising thestep of: collecting a specimen from the patient.
 35. The method of claim34 , wherein the specimen is collected following a period of fasting ofat least 6 hours.
 36. The method of claim 31 , further comprising thestep of, prior to administering a therapeutic agent to the patient:identifying the patient as a non-wound healing diabetic by the method ofclaim 1 .
 37. The method of claim 31 , further comprising the steps of:(c) comparing the level of the nitric oxide-related product in thespecimen with a threshold value which discriminates between woundhealing diabetics and non-wound healing diabetics; and (d) adjusting thetreatment according to the level of the nitric oxide-related product inthe specimen, wherein if the level is at or below the threshold valuethe administration of the therapeutic agent is increased, and if thelevel is above the threshold the administration of the therapeutic agentis not increased.
 38. The method of claim 37 , wherein if the level ofthe nitric oxide-related product following administration of thetherapeutic agent is at or below the threshold level, the method furthercomprises the step of: (e) repeating steps (a) through (d) until thelevel of nitric oxide-related product in a specimen from the patient isabove the threshold level.
 39. The method of claim 31 , wherein thetherapeutic agent is L-arginine or hyperbaric oxygen.
 40. A kit fordetermining whether a diabetic subject is a wound healing diabetic or anon-wound healing diabetic according to the method of claim 1 ,comprising one or more reagents for determining the level of a nitricoxide-related product in the specimen and instructions for using thereagents and interpreting the results.
 41. The kit of claim 40 , whereinthe specimen is urine, blood, or tissue.
 42. The kit of claim 40 ,wherein the nitric oxide-related product is nitrate, a derivative ofnitrate, nitrite, or a derivative of nitrite.
 43. The method of claim 40, wherein the nitric oxide-related product is nitric oxide,L-citrulline, cGMP, peroxynitrite, 3-nitrotyrosine, orL-dimethylarginine.